How Absolute Molar Mass can be Measured Using UHPLC SEC-MALS

Wyatt Technology Corporation has announced the availability of a new application note which outlines the combined use of the Wyatt μDAWN™ multi-angle light scattering (MALS) detector and the Optilab® UT-rEX™ refractive index (RI) detector to measure the absolute molecular weight (or molar mass) and size of eluting species in ultra high performance liquid chromatography (UHPLC). The study highlights how these instruments, engineered specifically for use with ultra-high performance size exclusion chromatography (UHP-SEC), overcome the limitations associated with conventional UHP-SEC for the characterization of size and composition of aggregates or fragments in a biomolecular sample.

Conventional SEC relies on reference standards to calibrate column elution time as a function of molar mass. However, these standards often do not accurately represent the conformation of the sample, or the sample may exhibit column interactions that change its elution properties relative to the standard. SEC-MALS with conventional HPLC is commonly utilized to determine molar masses independently of reference standards and elution time. The application note discusses how UHP-SEC-MALS provides improved resolution, higher throughput, less solvent and smaller sample consumption for analysis of precious biological samples in comparison to standard SEC-MALS. Light scattering detectors designed for standard HPLC have generally been incompatible with UHPLC. The powerful combination of the μDAWN and UT-rEX makes UHP-SEC-MALS a reality, resulting in faster and more efficient characterization of biomolecular samples.

The study demonstrates that the new detectors maintain the narrow peak widths typical of UHPLC while providing even more information than standard SEC-MALS thanks to the superb upstream resolution obtained with 300 mm UHPLC columns. The time difference between the two techniques clearly demonstrates how UHP-SEC-MALS results in much faster analyses. The application note also showcases how users can easily transition from existing HPLC methods to UHPLC while maintaining the same accurate, high-quality molecular weight data.

Geoffrey Wyatt, President of Wyatt Technology explains, “We are committed to helping raise standards in molecular analysis, and this new application note demonstrates the absolute quantification of molecules now possible through the use of the μDAWN and the Optilab UT-rEX. Our complete set of detectors enables researchers to enjoy the efficiency of UHPLC without sacrificing the data quality and the ability to measure absolute molar mass of a given protein in solution via MALS. We anticipate that this capability will help researchers achieve unprecedented levels of in-depth analysis for their UHPLC applications.”

With the introduction of the μDAWN, not only is it possible to quantify absolute molecular weight, users also have access to all the in-depth analyses available with the miniDAWN™ TREOS® for UHPLC. This range of instrumentation providing triple detection with UV, MALS, and RI also enables the application of Protein Conjugate Analysis to identify the amount of post-translational modification, associated detergent, drug-conjugate, or other additions to a polypeptide background.

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